Apparatus for preparing polynucleotides on a solid support

ABSTRACT

The present invention relates to an apparatus for preparing polynucleotides on a solid support in a reactor comprising a column containing an immobilized solid support that is functionalized for polynucleotide synthesis. The solid support can be porous, and can be functionalized with a nucleoside or with a universal synthesis of polynucleotides.

This application is a continuation of U.S. Ser. No. 09/119,653, filedJul. 21, 1998 now U.S. Pat. No. 6,623, 703, which is a divisional ofU.S. Ser. No. 08/358,556, filed Dec. 14, 1994, now U.S. Pat. No.5,869,643, which claims the benefit of priority of French applicationno. FR 93 15164, filed Dec. 16, 1993, now abandoned. Each of theforegoing applications/patents are incorporated herein in their entiretyby reference.

FIELD OF THE INVENTION

The present invention relates to a process for preparing polynucleotideson a solid support. The present invention also relates to a reactorcontaining a solid support and to a device including this reactor, whichare useful in the process for preparing polynucleotides according to theinvention.

BACKGROUND OF THE INVENTION

The synthesis of polynucleotides on a solid support is particularly usedin automated syntheses of DNA or RNA oligonucleotides. In the presentapplication, “polynucleotides” is understood to mean deoxyribonucleicacid or ribonucleic acid fragments or, more generally, polynucleotidesor oligonucleotides where the bases, internucleotide phosphate linkages,or alternatively the ribose rings of the bases, can be chemicallymodified in a known manner. This may be especially oligonucleotides withα or β anomers, oligonucleotides with inter-nucleotide linkage of thephosphorothioate or methyl phosphonate type, or alternativelyoligothionucleotides.

The principle for the chemical synthesis of nucleic acids on a solidsupport is nowadays widely described in the specialist literature, and anumber of apparatus are available on the market which performautomatically all or part of the synthesis steps. Among the chemicalroutes described, only the so-called phosphoramidites method (Carutherset al.: EP 0,035,719 BI) is up until now sufficiently efficient toenvisage the production of nucleic acids on an industrial scale.

The preparation of oligonucleotides or polynucleotides is carried out ina reactor containing a solid support and comprises the treatment of thesolid support such as an inorganic polymeric support by a series ofsuccessive steps, each of the series leading to the addition of a newnucleotide on the support. The series of successive steps, or synthesiscycles, are carried out as many times as is required by the manufactureof an oligonucleotide or a polynucleotide of desired length.

In a process for synthesizing polynucleotides on a solid support, thesolid support traditionally consists of glass beads of controlledporosity (CPG) or, more generally, of particles of a functionalizedinorganic or organic polymer.

The techniques conventionally used involve the use of eight differentreagents as solid supports, consisting of the said functionalizedinorganic or organic polymer linked to a nucleoside A, T, C, G or U,depending on whether the sequence to be prepared contains, as firstdeoxyribo- or ribonucleotide A, T, C, G or U. Manufacturers thereforesupply reactors in which one of these nucleosides has previously beenattached to the support. Depending on whether the sequence starts withA, T, C, G or U, the appropriate reactor is then chosen. The elongationof this first nucleoside then occurs in the 3′→5′, or 5′→3′ direction,using coupling reagents.

Numerous supports have already been described in the literature for thesolid phase synthesis of oligonucleotides.

There may be mentioned organic polymers such as polystyrene (NucleicAc.1980, volume 8), polyacrylamide acryloylmorpholide,polydimethylacrylamide polymerized on kieselguhr (Nucleic Ac. 9(7):691(1980)).

Other supports described are of inorganic nature, in particular based onsilica functionalized by a hydrocarbon radical carrying an NH₂ and/orCOOH group (J. Am. Chem., 105:61 (1983)), or the support based on silicafunctionalized by a 3-aminopropyltriethoxysilane group whose use inphosphite and phosporamidite synthesis for the preparation ofoligonucleotides was described for the first time in European Patent No.0,035,719.

There is known, from French Patent Application FR 93 08 498 andPCT/FR94/00842, a process for the solid phase synthesis ofoligonucleotides in which a so-called “universal” support is used, thatis to say a solid support which can be used regardless of the firstnucleotide of the RNA or DNA to be synthesized, regardless of the typeof monomeric reagent used during the synthesis, that is to sayregardless of the type of substitution on the phosphate group in 3′ orin 5′, depending on whether the synthesis is carried out in the 5′→3′ or3′→5′ direction.

In particular, the “universal nature” of the solid phase supports can beobtained by functionalization of the inorganic or organic polymer with ahydrocarbon radical containing glycol type groups in which an OH groupand a nucleophilic group are present in the vicinal position, that is tosay on two adjacent carbons, at the end of the hydrocarbon radical, itbeing possible for these two carbons to be optionally substituted byinert groups. “Inert group” is understood here to mean a group whichdoes not react under the conditions encountered during the various stepsof the synthesis on a solid support of nucleic acids according to theinvention.

In a specific embodiment, a process for synthesizing polynucleotidescomprises the following steps of:

1) condensing the OH group in 5′ or 3′ of the first nucleotide or of anoligonucleotide linked at its other 3′ or 5′ end to the said solidsupport, by means of a coupling agent, with the phosphate groupoptionally substituted respectively in position 3′ or 5′ of a monomericnucleotide reagent protected in 3′ and 5′;

2) oxidizing or sulfurizing the phosphite type internucleotide linkageobtained in step 1) into a phosphate linkage respectively;

3) deprotecting the 5′-O or 3′-O end of the product obtained in step 2);

4) repeating steps 1) to 3) as many times as there are nucleotides to beadded in order to synthesize the nucleic acid.

The above steps lead to an oligonucleotides linked to the solid support.The process comprises a final step of detaching the nucleic acid fromthe support and removing the groups for protecting the bases and, whereappropriate, the 2′-O positions of the nucleic acid.

In the techniques where the solid support is already linked to a firstnucleoside corresponding to the first nucleotide of the sequence to beprepared, before the start of the synthesis cycles, the said supportgenerally contains a protection in 5′ or 3′ of the said nucleoside. Inthis case, the synthesis cycle starts with a deprotection step in acidicmedium, in general a detritylation.

According to the variants used most commonly, the said monomericnucleotide reagent corresponds to the formula:

in which:

A represents H or an optionally protected hydroxyl group,

is a purine or pyrimidine base whose exocyclic amine functional group isoptionally protected,

C is a conventional protective group for the 5′-OH functional group,

x=0 or 1 with a) when x=1:

R₃ represents H and R₄ represents a negatively charged oxygen atom, or

R₃ is an oxygen atom and R₄ represents either an oxygen atom or anoxygen atom carrying a protecting group, and

b) when x=0, R₃ is an oxygen atom carrying a protecting group and R₄ iseither a hydrogen or a disubstituted amine group,

when x is equal to 1, R₃ is an oxygen atom and R₄ is an oxygen atom, themethod is in this case the so-called phosphodiester method; when R₄ isan oxygen atom carrying a protecting group, the method is in this casethe so-called phosphotriester method,

when x is equal to 1, R₃ is a hydrogen atom and 4 is a hydrogen atom andR₄ is a negatively charged oxygen atom, the method is in this case theso-called H-phosphonate method, and

when x is equal to 0, R₃ is an oxygen atom carrying a protecting groupand R₄ is either a halogen, the method is in this case the so-calledphosphite method and, when R₄ is a leaving group of the disubstitutedamine type, the method is in this case the so-called phosphoramiditemethod.

The steps of a cycle of synthesis by the phosphoramidite method areconventionally the following:

1) condensation of the 5′ terminal hydroxyl of a nucleoside or of anoligonucleotide covalently attached to the solid support with aphosphite type compound according to the reaction:

2) oxidation of the phosphite bond obtained to a phosphate according tothe reaction:

3) blocking of the hydroxyl groups of the unreacted nucleosides;

4) liberation of the 5′ terminal hydroxyl from the last nucleoside so asto generate an active site for the next synthesis cycle.

Each nucleotide is sequentially added to the support by repeating steps1 to 4. At the end of the synthesis, the oligonucleotide is separatedfrom the support and freed of its protecting groups by a controlledhydrolysis reaction.

Commercial synthesizers specialized in the synthesis of oligonucleotidesare designed so as to automatically carry out the synthesis stepsdescribed above. These synthesizers are generally composed of a reactorcontaining the solid support, a reagent mixer, one or several unit(s)for selecting the reagents and the vessels containing the said reagents.The synthesis steps are carried out by successively adding the selectedreagents to the reactor. Most often, the solid support is washed withacetonitrile after each step.

The solid support is not immobilized and does not occupy the wholevolume of the reactor but, in general, only half of the volume of thereactor, and in any case no more than three quarters, so as to allowadequate stirring of the solid phase.

The reactor, as used in commercial synthesizers, has the shape of avessel crossed by the flow of reagents which causes the stirring (orfluidization) of the solid support. It is indeed considered that thestirring of the solid phase is essential because it allows betterpenetration of the solvents and reagents into the pores of the solidphase generally consisting of porous beads or porous membranes (seeMethods in Molecular Biology: Protocols for Oligonucleotides andAnalogs—Synthesis and Properties—Edited by Sudhir Agrawal—1993—HumanaPress; Totowa, N.J., pages 442-444 and 454).

The mixer is situated upstream of the reactor, connected to it by apipeline. The units for selecting the reagents generally consist ofelectrovalves and allow selective opening of the inlets/outlets of thehydraulic circuit as well as the routes for the passage of the reagents.The cohesion of the hydraulic system is ensured by a series of suitablyconnected tubes or capillaries. It is recommended to install theequipment in an air-conditioned room at 20° C., the temperature at whichthe reactions are performed.

While the efficiency of the reactions is sufficient under theseconditions, the duration of the reactions and the required excess ofreagents in order to carry out good washing by successive dilutions arethe principal disadvantages of the synthesizers as described above. Inthis case, the consumption of reagents and the duration of the synthesesare substantially greater than the values calculated on the basis ofknown laws of organic synthesis. These disadvantages were attributed tothe limiting factor which the rate of diffusion of the reagents in thepores constitutes.

SUMMARY OF THE INVENTION

The aim of the invention is the improvement of the efficiencies ofexisting methods of synthesis of oligonucleotides.

The method according to the present invention consists in a modificationof organic synthesis apparatuses and parameters so as to improve theproductivity thereof. The improvements affect in particular the durationand the consumption of reagents necessary for carrying out a synthesis.

The subject of the present invention is also a reactor for preparingpolynucleotides according to a process of the invention, the reactorbeing in the form of a column containing a solid support through whichthe solutions of reagents and/or solvents are circulated, wherein thesolid phase constituting the solid support is immobilized in saidreactor such that said solutions migrate in the column and through saidsolid phase according to a frontal progression, the successive solutionsof each step of a synthesis cycle not mixing at all or very little.

In particular, the subject of the present invention is therefore areactor which is useful in a process for preparing polynucleotides on asolid support, which consists of a cylindrical column completely filledwith particles of porous materials constituting a solid support asdescribed above.

The subject of the present invention is finally a device for thesynthesis of polynucleotides on a solid support containing athermostatted reactor and a thermostatted collector which performs thecollection, the heating to the temperature of the reactor, then themixing of the reagents before their introduction into the reactor.

Other advantages and characteristics of the present invention willappear in the light of the detailed description below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a “LargeScale Synthesizer” of the invention.

FIG. 2 illustrates a “Multicolumn Synthesizer” of the invention.

DETAILED DESCRIPTION OF THE INVENTION

It was discovered according to the present invention that it is mainlythe geometry of the vessel-type reactor in which the free particles ofthe solid support are stirred according to the principle of“pseudoliquid” phase reaction which has numerous disadvantages.

It was also discovered that several other parameters are also calledinto question which considerably reduce the productivity of thesynthesizers:

the reagents used for the oxidation step (iodine solution,tetrahydrofuran, lutidine, water) are degraded over time, and theresulting reduction in reactivity rapidly induces a reduction in theefficiency of the synthesis;

the absence of thermostatting of the reactor and of the reagents leadsto the loss of reproducibility of the syntheses.

The hydraulic system itself often comprises unusable dead volumes. Thetime spent by the reagents in these dead volumes and in the mixingchamber, in particular during the activation of phosphoramidite by thecoupling agent, limits the reactivity of the transient species formed.

Accordingly, the present invention relates to a set of modificationsmade to the system described above which has the effect of improving theproductivity by reducing the consumption of reagents and the durationsof synthesis.

The essential characteristic of the process according to the presentinvention relates to the immobilization of the solid phase or to themethod of filling the reactor with the solid phase.

The subject of the present invention is indeed a process for preparingpolynucleotides on a solid support in a reactor in the form of a columnthrough which solutions of reagents and/or solvents are circulated,wherein the solid phase constituting said solid support is immobilizedin said reactor, and said solutions migrate in the column and throughthe solid phase according to a frontal progression, such that thesuccessive solutions from each step of a synthesis cycle do not mix.

The subject of the present invention is also a process for preparingpolynucleotides on a solid support in a reactor in the form of a columnthrough which the solutions of reagents and/or solvents are circulated,wherein the reactor is a column completely filled with particles of aporous material constituting said solid support.

In this embodiment where the solid support consists of free particles ofa porous material filling a column, the particles are also immobilized.The immobilization indeed results from the fact that by “completelyfilled column” there is understood here that the degree of filling withparticles and the density of distribution of the particles in the columnare such that the particles are sufficiently packed such that they canno longer move when a liquid crosses the column.

It may be only a portion of the column which is completely filledprovided that the particles of the solid support are maintained byappropriate means in said portion of the column.

In an advantageous embodiment, the column will have a cylindrical shape.It is thus proposed, for the first time, to use a “chromatographycolumn” type reactor, that is to say a column consisting of acylindrical tube completely filled with the solid phase.

This type of column, homogeneously filled with the solid support, offersseveral advantages: the various solutions which follow each other in thereactor do not mix, or very little, there is therefore no phenomenon ofdilution of the solutions by the previous reagent and the washingfollowing each step of the synthesis cycle is thereby greatlyfacilitated. According to the chromatography column principle, thesuccessive solutions introduced at one end of the column migrateaccording to a frontal progression, such that a solution “pushes” thepreceding one.

It was indeed observed that the solid phases conventionally used insolid phase oligonucleotides synthesis are such that the affinity andthe physicochemical interactions of the various reagents and solventsused are negligible and do not interfere with their diffusion throughthe solid phase other than by the desired chemical reactions.

It should be mentioned that, of course, in the interface region betweenthe solutions, slight mixing may occur locally between the solutionswhen the circulation rates of the solutions are high. However, thismixing, which is localized at the interface, is in no way comparablewith what is observed in prior processes for which the washes were basedon the principle of dilution by mixing successive solutions.

Contrary to the conventional model, these reactors therefore make itpossible to substantially reduce the volumes of reagents required. Theuseful flow rates are adjusted as a function of the diffusion constantsand the kinetics of the reactions called into play. The duration of thesynthesis cycles is thereby considerably reduced. For each type ofparticle, depending on its porosity, the flow rate of the reagentsthrough the column should not exceed a certain value above which thereagents no longer diffuse inside the pores. This is a well knownphenomenon in chromatography.

In addition, within the reactor and regardless of its size, theconditions are locally always identical. The system can therefore beapplied to all scales of synthesis.

In particular, there may be mentioned materials consisting of inorganicpolymers, especially glass, silica, metal oxides, or of organicpolymers, especially cellulose, or optionally substituted polystyrene.

Preferably, the polymer is an inorganic polymer prepared based on glassor silica, especially a silica gel.

The said particles may consist of microbeads or agglomeratedmicrofibers.

Preferably, in the reactor according to the present invention, there maybe used as particles of the solid support porous microbeads, therebyproviding the largest functionalized surface area in terms of weight ofnucleosides linked to the polymer per weight of polymers. The size ofthe beads may range from 5 μm to 500 μm, more generally from 40 μm to 70μm. The pores may optionally pass right through the microbeads. Thediameter of the pores may range between 200 Å and 10,000 Å. Two poresizes may be used, with wide pores (about 10,000 Å) and smaller pores(300 Å). This type of pore of variable size favors the diffusion of thesolutions through the solid phase.

The reactor according to the present invention makes it possible to bestexploit known laws of diffusion and kinetics of bimolecular chemicalreactions according to which the reagents in solution diffuse veryrapidly into the pores of the solid support and the kinetics ofcondensation of the free nucleoside with the 5′OH end of theoligonucleotides attached to the support is practically instantaneous.

The regulation of the temperature of the reactor and the reagents isanother essential characteristic of the present invention which, so far,does not exist in any synthesizer on the market.

The working temperature is one of the parameters which directly andsimultaneously influence the kinetics of the chemical reactions calledinto play, but also the stability of the reagents and the viscosity ofthe solutions. Contrary to all the prejudice which claims that thesynthesis of the oligonucleotides decreases the efficiency above 30° C.,it is recommended according to the present invention to thermostat thereactor at an optimal temperature of 45° C. The regulation of thetemperature of the system makes it possible to act on the kinetics ofthe chemical reactions called into play as well as on the viscosity ofthe solutions used.

Advantageously, the reagents, that is to say the coupling reagents suchas tetrazole, and the monomeric reagents, on the one hand, and/or thevarious reagents of the oxidizing solution, on the other hand, are mixedimmediately upstream of the reactor, or optionally, just upstream of asystem of distribution of the reagents in a multicolumn system. Theunusable dead volumes are thereby reduced.

In particular, the reagents are heated before introducing them into thereactor and, where appropriate, before mixing them.

Indeed, if the reagents are heated only after mixing them, a mutualinactivation and a loss of reactivity on the column are observed. Moreprecisely, early mixing (too far from the reactor) of the reagents canlead to undesirable side reactions. In particular, the mixing of thetetrazole (weak acid) and the phosphoramidites can result in thedetritylation of part of the monomers. The phosphoramidites can thenreact with each other. This type of reaction leads to the formation ofhomopolymers which may or may not react with the oligonucleotideattached to the support. The formation of oligonucleotides which arelonger than desired can be observed in this case. Whatever the case, thesynthesis yield is always thereby reduced. Of course, these phenomenaare accentuated by heating. It is therefore advisable to heat thereagents before mixing them.

In the reactors used before the present invention, above 30° C., thereagents become rapidly degraded. But, by means of the reactor accordingto the present invention, which favors the kinetics of the reactions, itis possible to increase the temperature up to 90° C. (between 20 and 90°C.), preferably the temperature of the reactor is adjusted between 30and 60° C., especially to 45° C.

Advantageously, the deprotection step in acidic medium is performed withTFA (trifluoroacetic acid) in dichloromethane. A 1% v/v solution oftrifluoroacetic acid in extra dry dichloroethane is especially usedduring the step for liberation of the terminal 5′ hydroxyl functionalgroup (step 4) by replacing the standard solution (2% dichloroaceticacid in dichloromethane). This recommendation makes it possible inparticular to get rid of the possible phenomena of boiling ofdichloromethane when the working temperature exceeds 40° C.

The mixing of acetic acid and pyridine causes an increase in thetemperatures from 40° to 60° C., which facilitates the maintenance ofthe temperature at said temperatures.

By virtue of the very high coupling yields (probably greater than 99%)of the process according to the invention, the step of blocking (or“capping”) the unreacted 5′-OH functional groups (step 3) proves, inmost cases, not to be very efficient, or even completely pointless andcan therefore be omitted. Thus, no accumulation of synthetic fragmentsof size n-1 is observed as would be expected if no capping step isperformed. Probably, when the efficiency of the reactions is optimal,the unreacted species are permanently inaccessible.

According to another characteristic of the present invention, theoxidation step is performed with iodine in acetic acid and pyridine.Thus, in place of the conventional oxidation solution, two reagents areused separately, one consisting for example of an iodine solutionsaturated with glacial acetic acid, the other being ultrapure pyridine.These reagents are advantageous in that, at the oxidation step (step 2above), these two solutions can be mixed in stoichiometric quantities atthe inlet of the reactor. The mixture thus formed is injected into thereactor and instantaneously and quantitatively ensures the oxidation ofthe newly formed internucleotide bond. Thus, advantage is taken of thestability of the two separate solutions by indefinitely guaranteeing thereproducibility of the oxidation step.

Two oligonucleotides or polynucleotide synthesizers, designed on thebasis of the technical improvements described above, were produced. Theperformances and the productivity of these two apparatus are therebysubstantially increased compared to apparatus on the market.

The first synthesizer was designed to synthesize quantities ofoligonucleotides between 50 μmol and 1 mmol. These performances cantherefore be compared to those of commercial synthesizers which arepresent on the market and are capable of working at equivalent synthesisscales. This apparatus is called “LargeScale Synthesizer”. It isrepresented in FIG. 1.

The function of the second apparatus is to synthesize, in parallel, 24different oligonucleotides or polynucleotides. The scale of eachsynthesis is between 0.05 and 1 μmol. So far, this apparatus has noequivalent. Its productivity can be compared to that of 6 synthesizers—4columns on the market. It is called “Multicolumn Synthesizer”. It isrepresented in FIG. 2.

These two apparatus comprise:

the vessels [(1) to (10)] containing the reagents, each comprising asyringe type positive displacement pumping module [(11) to (20)];

a thermostatted collector [(21)] whose attributes are the collection,the heating and the regulation of the temperature and the mixing of themoving reagents.

The “LargeScale Synthesizer” is equipped with a “filled column” typereactor whose dimensions are adjusted as a function of the desiredsynthesis scale [(22)].

The “Multicolumn Synthesizer” is equipped with 24 microcolumns [(22)] ofthe same type as the “Large-Scale Synthesizer” but whose dimensions makeit possible to work at the scales 0.05 μmol, 0.1 μmol, 0.2 μmol and 1μmol. Between the collector and the reactors is a distributor [(23)]responsible for equally distributing the reagents between the 24columns.

Finally, a block composed of 24 electrovalves, individually controlledat each column, makes it possible to select, at each synthesis step, thereactors “in service”.

The volumes, the flow rates and the waiting times are under the controlof a computer. The program controls the correct sequence and theexecution of the cycles in conformity with the sequences to besynthesized.

In order to guarantee local conditions which are always identicalregardless of the number -of reactors in service, the “MulticolumnSynthesizer” functions according to an interactive mode. Thus, thevolumes and the flow rates of each of the reagents are adjusted, at eachinstant, to the number of syntheses in progress.

A—Course of a Synthesis with Phosphoramidite in the LargeScaleSynthesizer

The synthesis scale is chosen at the time the program is initiated.

Each synthesis starts with an initialization cycle which makes itpossible to wash and thermostat the reactor to the working temperature.

The program comprises five synthesis cycles corresponding to each of thefour bases and to the possible modified bases.

The synthesis cycles can be broken down into synthesis steps orprocedures which follow one another in the following chronologicalorder:

1) Detritylation: liberation of the hydroxyl in position 5′ of thenucleoside or of the oligonucleotide covalently attached to the solidsupport,

2) First washing;

3) Addition of the base: condensation of the phosphoramidite typemonomer with the free 5′ terminal hydroxyl of the nucleoside or of theoligonucleotide attached to the solid support. Formation ofinternucleotide bond;

4) Second washing;

5) Oxidation of the previously formed phosphite type internucleotidebond into phosphate;

6) Third washing.

These cycles are repeated as many times as is required by theoligonucleotide synthesis or the synthesis of a polynucleotide ofdesired length.

B—Course of a Synthesis in the Multicolumn Synthesizer

The synthesis scale is chosen at the time the program is initiated.

Each synthesis starts with an initialization cycle which makes itpossible to wash and thermostat the reactors to the working temperature.

There is only one synthesis cycle which is repeated as many times as isrequired by the manufacture of the 24 oligo- or polynucleotides.

The cycle can be broken down in the following manner:

a) selection of the columns to be detritylated.

1) Detritylation: liberation of the hydroxyl in position 5′ of thenucleoside or of the oligonucleotide covalently attached to the solidsupport.

2) First washing:

b1) Selection of the columns which should receive the A base.

3-1) Addition of the A base: condensation of the phosphoramidite type Amonomer with the free 5′ terminal hydroxyl nucleoside or of theoligonucleotide attached to the solid support. Formation of theinternucleotide bond.

b2) Selection of the columns which should receive the G base.

3-2) Addition of the G base: condensation of the phosphoramidite type Gmonomer with the free 5′ terminal hydroxyl of the nucleoside or of theoligonucleotide attached to the solid support. Formation of theinternucleotide bond.

b3) Selection of the columns which should receive the T base.

3—3) Addition of the T base: condensation of the phosphoramidite type Tmonomer with the free 5′ terminal hydroxyl of the nucleoside or of theoligonucleotide attached to the solid support. Formation of theinternucleotide bond.

b-4) Selection of the columns which should receive the C base.

3-4) Addition of the C base: condensation of the phosphoramidite type Cmonomer with the free 5′ terminal hydroxyl of the nucleoside or of theoligonucleotide attached to the solid support. Formation of theinternucleotide bond.

c) Selection of the oxidizing columns.

4) Second washing.

5) Oxidation of the previously formed phosphite type internucleotidebond into phosphate.

6) Third washing.

DESCRIPTION OF THE STEPS MENTIONED ABOVE

Each step can be described with reference to the diagrams provided forthe synthesizers (FIGS. 1 and 2).

1) Detritylation: reagent no. 10, preferably a 1% v/v solution oftrifluoroacetic acid in extra dry dichloroethane, is pushed through thereactor by means of the syringe module no. 10.

 The volume necessary is between 2 and 10 times the volume of the emptyreactor.

 The flow rate in the column is less than 500 cm/min. This flow rateunit in cm/min in fact represents the linear speed of the solutionsinside the column and is therefore independent of the diameter of thecolumn. The volume flow rates should be adjusted as a function of thediameter of the reactor. In order to change the size of the reactorwhile locally preserving the synthesis conditions, it is sufficient tomodify the volume flow rate while ensuring that the linear speed insidethe reactor, given in cm/min, does not change.

 The flow rates which are used are compatible with the diffusionconstants in the pores, that is to say that the linear speed of thesolutions inside a column is not greater than the speed of diffusionthrough the pores. In the opposite case, there would be a low efficiencyof the reagents and of the washes resulting in a decrease in thesynthesis yields.

 The total reaction time is between 10 and 120 seconds.

2) First washing: the reagent no. 1, preferably extra dry acetonitrile,is pushed through the reactor by means of the syringe module no. 1.

 The volume required is between 2 and 10 times the volume of the emptyreactor.

 The flow rate in the column is less than 500 cm/min.

3) Coupling: the reagents no. 3, or no. 4, or no. 5 or no. 6, or no. 7,and no. 2, respectively a 0.1 M solution of each monomer and a 0.45%solution of tetrazole in acetonitrile, are conjointly pushed through thereactor by means of the syringe modules no. 3, or no. 4, or no. 5, orno. 6, or no. 7, and no. 2 respectively.

 The ratios of the volumes and the flow rates of the reagents no. 3, orno. 4, or no. 5, or no. 6, or no. 7, and no. 2 may be different from 1.

 The total volume required is between 2 and 10 times the volume of theempty reactor.

 The overall flow rate in the column is less than 500 cm/min.

 The reaction time is less than 1 minute.

4) Second washing: the reagent no. 1, preferably extra dry acetonitrile,is pushed through the reactor by means of the syringe module no. 1.

 The volume required is between 0.5 and 5 times the volume of the emptyreactor.

 The flow rate in the column is less than 500 cm/min.

5) Oxidation: the reagents no. 8 and no. 9, respectively a saturatedsolution of iodine in glacial acetic acid and ultrapure pyridine, areconjointly pushed through the reactor by means of the syringe modulesno. 8 and no. 9 respectively.

 The ratios of the volumes and flow rates of the reagents no. 8 and no.9 are equal to 1.

 The total volume required is between 1 and 5 times the volume of theempty reactor.

 The overall flow rate in the column is less than 500 cm/min.

 The reaction time is less than 30 seconds.

6) Third washing: the reagent no. 1, preferably extra dry acetonitrile,is pushed through the reactor by means of the syringe module no. 1.

 The volume required is between 1 and 5 times the volume of the emptyreactor.

 The flow rate in the column is less than 500 cm/min.

The following exemplary embodiment sizes serve to illustrate the processaccording to the invention.

EXAMPLES Example 1 Synthesis of an Oligonucleotide, at the 30 μmolscale, with the aid of the LargeScale Synthesizer

The apparatus used is that previously described.

The reactor used is a cylindrical glass column, of diameter 10 mm andheight 25 mm.

The working temperature is 45° C.

The synthesis cycles are detailed in Table 1.

An oligodeoxynucleotide, 18 bases long, whose sequence is: d(ACG TTC CTCCTG CGG GAA) is synthesized under these conditions.

The reactor is carefully filled with 0.66 g of CPG 500 Å (CPG INC.,USA), “derivatized” by the first A nucleoside. The capacity of thesupport is 45 μmol/g (density of 3 ml/g).

The synthesis scale is 30 μmol.

After a step of washing with acetonitrile, the synthesis cycle asdescribed in Table 1 is performed 17 times.

Under these conditions, the oligonucleotide, of the desired length,retains the transient dimethoxytrityl group at the 5′ terminal position.

The oligonucleotide is separated from the CPG and liberated from thepermanent protecting groups by an appropriate treatment of the support,“derivatized” according to the method described above, with 10 ml of a30% aqueous solution of ammonium hydroxide, for 16 hours at 55° C.

After adding 40 ml of absolute ethanol and 1 ml of a 3M aqueous solutionof sodium acetate, the oligonucleotide is left to precipitate for twohours at 0° C.

The precipitate is then filtered on a 1.2 μ lopridyne membrane (PALLS.A., FRANCE) and resolubilized in 10 ml of water.

After reading the optical density at 260 mm, 4000 O.D.U., that is to say120 mg of crude synthesis mixture, are obtained.

The purity of the oligonucleotide of the desired length, estimated byHPLC on a reversed-phase column, is 88%.

Example 2 Synthesis of an Oligonucleotide, at the 77 μmol Scale with theAid of the LargeScale Synthesizer

The apparatus used is the same as for the preceding example.

The reactor used is a cylindrical glass column, of diameter 10 mm andheight 25 mm.

The working temperature is 45° C.

The synthesis cycles are detailed in Table 2.

An oligodeoxynucleotide, 18 base in length, whose sequence is: d(TTC CGCCAG GAG GAA CGT) was synthesized under these conditions.

The reactor is carefully filled with 0.66 g of High-Loaded CPG 500 Å,“derivatized” by the first T nucleoside. The capacity of the support is110 μmol/g, its density 3 ml/g (MILLIPORE S.A., FRANCE).

The synthesis scale is 77 μmol.

After a step of washing with acetonitrile, we performed the synthesiscycle as described in Table 2 17 times.

Under these conditions, the oligonucleotide, of the desired length,retains the transient dimethoxytrityl group at the 5′ terminal position.

The conditions for deprotection and recovery of the oligonucleotide arethe same as those described in the preceding example.

After reading the optical density at 260 nm, 8,500 O.D.U., that is tosay 255 mg of crude synthesis mixture, are obtained.

The purity of the oligonucleotides of the third length, estimated byHPLC on a reversed-phase column, is 84%.

Example 3 Synthesis of an Oligonucleotide, at the 100 μmol Scale, withthe Aid of the LargeScale Synthesizer

The apparatus used is the same as for the previous example.

The reactor used is a cylindrical glass column, of diameter 15 mm andheight 34 mm.

The working temperature is 45° C.

The synthesis cycles are detailed in Table 3.

An oligonucleotide, 56 bases in length, whose sequence is d(TAA CCA CACTTT TTG TGT GGT TAA TGA TCT ACA GTT ATT TTT TAA CTG TAG ATC AT) issynthesized under these conditions.

The reactor is carefully filled with 2 g of CPG 500 Å, “derivatized” bythe first T nucleoside. The capacity of the support is 50 μmol/g, itsdensity 3 ml/g (MILLIPORE S.A., FRANCE).

The synthesis scale is 100 μmol. After a step of washing withacetonitrile, we performed the synthesis cycle as is described in Table3 55 times.

Under these conditions, the oligonucleotide, of the desired length,retains the transient dimethoxytrityl group at the 5′ terminal position.

The conditions for deprotection and recovery of the oligonucleotide arethe same as those described in the previous examples.

After reading the optical density at 260 nm, 31,000 O.D.U., that is tosay 930 mg of crude synthesis mixture, are obtained.

The purity of the oligonucleotide of length 56 mers, estimated by HPLCon a reversed-phase column, is 61%.

Example 4 Simultaneous Syntheses of 24 Different Oligonucleotides withthe Aid of the Multicolumn Synthesizer

The reactors are metallic micro columns of diameter 1.5 mm and height 6mm.

The working temperature is 50° C.

The synthesis cycles are detailed in Table 4.

We synthesized, under these conditions, the oligodeoxynucleotides whosesequences are given in Table 5.

The reactors are evenly filled with 2 mg of universal CPG 500 Åcontaining an epoxide group (prepared according to Example 1 of patentapplication FR 93 08 498). The capacity of the support is 50 μmol/g(GENSET S.A., FRANCE) (density: 3 ml/g).

The synthesis scale is 0.1 μmol.

After a step of washing with acetonitrile, we performed the operationsdescribed in Table 4 as many times as is required by the synthesis ofthe 24 oligodeoxynucleotides of Table 5.

The conditions for deprotection and recovery of the oligonucleotide arethe same as those described in the preceding examples.

After reading the optical density at 260 nm, 11 O.D.U. are obtained onaverage from each of the oligonucleotides, that is to say 0.33 mg. Thepurity of the oligonucleotides, estimated by HPLC, on a reversed-phasecolumn for the 5′ O-Trityl oligodeoxynucleotides, and on an anionexchange column for the 5′OH oligonucleotides, exceeds 84%.

Comparative Example 5

Table 6 below presents the characteristics of the syntheses carried outwith fluidized bed reactors marketed by APPLIED BIOSYSTEMS and MILLIPOREas described in the literature.

The synthesizers used are the following:

APPLIED SYNTHESIZER, model 394:

reactor in the form of a column:

diameter 5 mm

height 6 mm

volume 0.11 ml

Solid support: CPG 500 Å (CPG INC., USA)

capacity: 30 μmol/g

density: 3 ml/g

For a synthesis at the 0.2 μmol scale:

6.7 mg of CPG, that is to say a volume of 0.02 ml and a degree

of filling of 20%.

MILLIPORE SYNTHESIZER, model 8800:

Reactor in the form of a 225 ml vessel which can be used with 1 to 15 gof synthesis of CPG.

For our examples:

“STANDARD” method for 100 μmol

solid support: CPG 500 Å (MILLIPORE S.A., FRANCE)

capacity: 30 μmol/g

density: 3 ml/g

For 100 μmol: 3.33 g of CPG that is to say 10 ml of phase and a degreeof filling of 4.5%.

“IMPROVED” method for 100 μmol

solid support: HIGH-LOADED CPG 500 Å (MILLIPORE S.A., FRANCE)

capacity: 100 μmol/g

density: 3 ml/g

For 100 μmol: g of CPG that is to say 3 ml of phase and a degree offilling of 1.3%.

In comparison with the results described in Examples 1 to 4, thedecrease in the duration of synthesis and the quantities of reagents isconsiderable. For the 100 μmol:

with the process according to the invention (Example 3 and Table 3), 118ml of reagents and solvents are used and the duration of synthesis is 3minutes per synthesis cycle;

with a MILLIPORE column, 500 ml of reagents and solvents are used, andthe duration of synthesis is 20 minutes per cycle.

TABLE 1 VOL- IND. FLOW RATE SOLVENTS OR REAGENTS VESSEL NO. UME RATETOTAL FLOW TIME Dichloroethane, TFA 1% v/v 10 20 ml 72 ml/min 17 secWaiting time 20 sec Acetonitrile 1 8 ml 72 ml/min  7 sec 0.45M Tetrazolein acetonitrile 2 3 ml 60 ml/min  3 sec 0.45 Tetrazole in acetonitrile 26 ml 36 ml/min plus 48 ml/min 10 sec 0.1M monomer in acetonitrile 3, 4,5, 6 or 7 2 ml 12 ml/min Waiting time 20 sec 0.45M Tetrazole inacetonitrile 2 1 ml 60 ml/min  1 sec Acetonitrile 1 2 ml 60 ml/min  2sec Iodine sat. with Acetic Acid 8 2.5 ml 30 ml/min plus 60 ml/min  5sec Pyridine 9 2.5 ml 30 ml/min Acetonitrile 1 10 ml 60 ml/min 10 secTOTAL 57 ml  1 min 35 sec

TABLE 2 IND. FLOW VOL- RATE SOLVENTS OR REAGENTS VESSEL NO. UME RATETOTAL FLOW TIME Dichloroethane, TFA 1% v/v 10 20 ml 72 ml/min 17 secWaiting time 30 sec Acetonitrile 1 8 ml 72 ml/min  7 sec 0.45M Tetrazolein acetonitrile 2 3 ml 60 ml/min  3 sec 0.45 Tetrazole in acetonitrile 26 ml 36 ml/min plus 48.6 ml/min 10 sec 0.1M monomer in acetonitrile 3,4, 5, 6 or 7 2.1 ml 12.6 ml/min Waiting time 40 sec 0.45M Tetrazole inacetonitrile 2 1 ml 60 ml/min  1 sec Acetonitrile 1 2 ml 60 ml/min  2sec Iodine sat. with Acetic Acid 8 2.5 ml 30 ml/min plus 60 ml/min 5 secPyridine 9 2.5 ml 30 ml/min Acetonitrile 1 10 ml 60 ml/min 10 sec TOTAL57.1 ml  2 min 05 sec

TABLE 3 VOL- IND. FLOW RATE SOLVENTS OR REAGENTS VESSEL NO. UME RATETOTAL FLOW TIME Dichloroethane, TFA 1% v/v 10 40 ml 84 ml/min 30 secWaiting time 30 sec Acetonitrile 1 14 ml 72 ml/min 15 sec 0.45MTetrazole in acetonitrile 2 6 ml 60 ml/min  6 sec 0.45 Tetrazole inacetonitrile 2 14 ml 36 ml/min plus 48 ml/min 25 sec 0.1M monomer inacetonitrile 3, 4, 5, 6 or 7 5 ml 12 ml/min Waiting time 45 sec 0.45MTetrazole in acetonitrile 2 3 ml 60 ml/min  3 sec Acetonitrile 1 6 ml 60ml/min  6 sec Iodine sat. with Acetic Acid 8 5 ml 30 ml/min plus 60ml/min 10 sec Pyridine 9 5 ml 30 ml/min Acetonitrile 1 20 ml 60 ml/min15 sec TOTAL 118 ml  3 min 05 sec

TABLE 4 Dichloro- 0.45M tetrazole Sol. of ethanel Aceto- 0.45M 0.1M0.45M Aceto- Iodine Aceto- TOTAL TIME/ % v/v TFA nitrile TetrazoleMonomers Tetrazole nitrile sat. Pyridine nitrile VOLUME CYCLE STEPDetritylation Wash 1 Coupling Coupling Wash 2 Wash 2 Oxidation Wash 3(ml) (min) VESSEL 10 1 2 3, 4, 5, 6 or 7 2 1 8 and 9 1 NO. and 2 1VOLUME 0.8 0.4 0.0375 0.08 0.025 0.4 0.1 0.4 2.2425 COLUMNS (ml) FLOWRATE 6 4.8 2.25 2.94 1.5 4.8 3 4.8 0.48 (ml/min) 2 VOLUME 1.2 0.6 0.0750.16 0.05 0.6 0.15 0.6 3.435 COLUMNS (ml) FLOW RATE 9 7.2 4.5 2.94 3 7.23.15 7.2 0.52 (ml/min) 3 VOLUME 1.6 0.8 0.1125 0.24 0.075 0.8 0.2 0.84.6275 COLUMNS (ml) FLOW RATE 12 9.6 6.75 5.88 4.5 9.6 3.3 9.6 0.52(ml/min) 4 VOLUME 2 1 0.15 0.32 0.1 1 0.25 1 5.82 COLUMNS (ml) FLOW RATE15 12 9 8.82 6 12 3.45 12 0.53 (ml/min) 5 VOLUME 2.4 1.2 0.1875 0.40.125 1.2 0.3 1.2 7.0125 COLUMNS (ml) FLOW RATE 18 14.4 11.25 11.76 7.514.4 3.6 14.4 0.53 (ml/min) 6 VOLUME 2.8 1.4 0.225 0.48 0.15 1.4 0.351.4 8.205 COLUMNS (ml) FLOW RATE 21 16.8 13.5 14.7 9 16.8 3.75 16.8 0.54(ml/min) 7 VOLUME 3.2 1.6 0.2625 0.56 0.175 1.6 0.4 1.6 9.3975 COLUMNS(ml) FLOW RATE 24 19.2 15.75 17.64 10.5 19.2 3.9 19.2 0.55 (ml/min) 8VOLUME 3.6 1.8 0.3 0.64 0.2 1.8 0.45 1.8 10.59 COLUMNS (ml) FLOW RATE 2721.6 18 20.58 12 21.6 4.05 21.6 0.56 (ml/min) 9 VOLUME 4 2 0.3375 0.720.225 2 0.5 2 11.7825 COLUMNS (ml) FLOW RATE 30 24 20.25 23.52 13.5 244.2 24 0.56 (ml/min) 10 VOLUME 4.4 2.2 0.375 0.8 0.25 2.2 0.55 2.212.975 COLUMNS (ml) FLOW RATE 33 26.4 22.5 26.46 15 26.4 4.35 26.4 0.57(ml/min) 11 VOLUME 4.8 2.4 0.4125 0.88 0.275 2.4 0.6 2.4 14.1675 COLUMNS(ml) FLOW RATE 36 28.8 24.75 29.4 16.5 28.8 4.5 28.8 0.58 (ml/min) 12VOLUME 5.2 2.6 0.45 0.96 0.3 2.6 0.65 2.6 15.36 COLUMNS (ml) FLOW RATE39 31.2 27 32.34 18 31.2 4.65 31.2 0.59 (ml/min) 13 VOLUME 5.6 2.80.4875 1.04 0.325 2.8 0.7 2.8 16.5525 COLUMNS (ml) FLOW RATE 42 33.629.25 35.28 19.5 33.6 4.8 33.6 0.59 (ml/min) 14 VOLUME 6 3 0.525 1.120.35 3 0.75 3 17.745 COLUMNS (ml) FLOW RATE 45 36 31.5 38.22 21 36 4.9536 0.60 (ml/min) 15 VOLUME 6.4 3.2 0.5625 1.2 0.375 3.2 0.8 3.2 18.9375COLUMNS (ml) FLOW RATE 48 38.4 33.75 41.16 22.5 38.4 5.1 38.4 0.60(ml/min) 16 VOLUME 6.8 3.4 0.6 1.28 0.4 3.4 0.85 3.4 20.13 COLUMNS (ml)FLOW RATE 51 40.8 36 44.1 24 40.8 5.25 40.8 0.61 (ml/min) 17 VOLUME 7.23.6 0.6375 1.36 0.425 3.6 0.9 3.6 21.3225 COLUMNS (ml) FLOW RATE 54 43.238.25 47.04 25.5 43.2 5.4 43.2 0.61 (ml/min) 18 VOLUME 7.6 3.8 0.6751.44 0.45 3.8 0.95 3.8 22.515 COLUMNS (ml) FLOW RATE 57 45.6 40.5 49.9827 45.6 5.55 45.6 0.62 (ml/min) 19 VOLUME 8 4 0.7125 1.52 0.475 4 1 423.7075 COLUMNS (ml) FLOW RATE 60 48 42.75 52.92 28.5 48 5.7 48 0.62(ml/min) 20 VOLUME 8.4 4.2 0.75 1.6 0.5 4.2 1.05 4.2 24.9 COLUMNS (ml)FLOW RATE 63 50.4 45 55.86 30 50.4 5.85 50.4 0.62 (ml/min) 21 VOLUME 8.84.4 0.7875 1.68 0.525 4.4 1.1 4.4 26.0925 COLUMNS (ml) FLOW RATE 66 52.847.25 58.8 31.5 52.8 6 52.8 0.63 (ml/min) 22 VOLUME 9.2 4.6 0.825 1.760.55 4.6 1.15 4.6 27.285 COLUMNS (ml) FLOW RATE 69 55.2 49.5 61.74 3355.2 6.15 55.2 0.63 (ml/min) 23 VOLUME 9.6 4.8 0.8625 1.84 0.575 4.8 1.24.8 28.4775 COLUMNS (ml) FLOW RATE 72 57.6 51.75 64.68 34.5 57.6 6.357.6 0.64 (ml/min) 24 VOLUME 10 5 0.9 1.92 0.6 5 1.25 5 29.67 COLUMNS(ml) FLOW RATE 75 60 54 67.62 36 60 6.45 60 (ml/min)

TABLE 5 COLUMN 5′ No NO. OLIGO Trityl bases 5′-3′ SEQUENCE 0 OLIGO1 YES12 TTT TTT TTT TTT 1 OLIG02 NO 12 TTT TTT TTT TTT 2 OLIG03 YES 15 TTTTTT TTT TTT TTT 3 OLIG04 NO 15 TTT TTT TTT TTT TTT 4 OLIG05 YES 18 TTTTTT TTT TTT TTT TTT 5 OLIG06 NO 18 TTT TTT TTT TTT TTT TTT 6 OLIG07 YES12 AAA AAA AAA AAA 7 OLIG08 NO 12 AAA AAA AAA AAA 8 OLIG09 YES 15 AAAAAA AAA AAA AAA 9 OLIGO10 NO 15 AAA AAA AAA AAA AAA 10 OLIGO11 YES 18AAA AAA AAA AAA AAA AAA 11 OLIG012 NO 18 AAA AAA AAA AAA AAA AAA 12OLIG013 YES 12 CCC CCC CCC CCC 13 OLIG014 NO 12 CCC CCC CCC CCC 14OLIG015 YES 15 CCC CCC CCC CCC CCC 15 OLIG016 NO 15 CCC CCC CCC CCC CCC16 OLIG017 YES 18 CCC CCC CCC CCC CCC CCC 17 OLIG018 NO 18 CCC CCC CCCCCC CCC CCC 18 OLIG019 YES 12 AGT CAG TCA GTC 19 OLIG020 NO 12 AGT CAGTCA GTC 20 OLIG021 YES 15 AGT CAG TCA GTC AGT 21 OLIG022 NO 15 AGT CAGTCA GTC AGT 22 OLIG023 YES 18 AGT CAG TCA GTC AGT CAG 23 OLIG024 NO 18AGT CAG TCA GTC AGT CAG

TABLE 6 MILLIPORE LARGE- MILLIPORE SYNTHESIZERS ABI 394 SCALE LARGE-CHARACTERISTICS 4 COLUMNS 100 μmol SCALE SYNTHESIS SCALE 0.2 μmol STAND-100 μmol METHOD STANDARD* ARD** IMPROVED** REAGENTS (ml) (ml) (ml)MONOMERS 0.1M 0.110 5 2.62 TETRAZOLE 0.400 20.3 19 SOLUTION CAP A 0.2909 6.25 (acetic anhydride) CAP B 0.260 11.6 8.65 (N-methylimidazole)DEBLOCK (TCA/DCM) 1.100 84.3 68.75 IODINE SOLUTION 0.350 40 33ACETONITRILE 8.000 376 286.25 TOTAL 10,510 546.2 424.52 TIME PER CYCLE 6min 20 min 22 min *APPLIED BIOSYSTEMS Instruction manual for the ABI 394synthesizer **N.D. SINHA, S. FRY; 3rd CAMBRIDGE SYMPOSIUMOligonucleotides & Analogues, 5-8 Sep. 1992 POSTER COMMUNICATION

31 18 base pairs nucleic acid single linear DNA (genomic) NO NON-terminal CDS 1..18 1 ACG TTC CTC CTG CGG GAA 18 Thr Phe Leu Leu ArgGlu 1 5 6 amino acids amino acid linear protein 2 Thr Phe Leu Leu ArgGlu 1 5 18 base pairs nucleic acid single linear DNA (genomic) NO NON-terminal CDS 1..18 3 TTC CGC CAG GAG GAA CGT 18 Phe Arg Gln Glu GluArg 1 5 6 amino acids amino acid linear protein 4 Phe Arg Gln Glu GluArg 1 5 56 base pairs nucleic acid single linear DNA (genomic) NO NON-terminal CDS 1..56 5 TAA CCA CAC TTT TTG TGT GGT TAA TGA TCT ACA GTTATT TTT TAA CTG 48 * Pro His Phe Leu Cys Gly * * Ser Thr Val Ile Phe *Leu 1 5 10 15 TAG ATC AT 56 * Ile 6 amino acids amino acid linearprotein 6 Pro His Phe Leu Cys Gly 1 5 5 amino acids amino acid linearprotein 7 Ser Thr Val Ile Phe 1 5 12 base pairs nucleic acid singlelinear DNA (genomic) NO NO N-terminal CDS 1..12 8 TTT TTT TTT TTT 12 PhePhe Phe Phe 1 4 amino acids amino acid linear protein 9 Phe Phe Phe Phe1 15 base pairs nucleic acid single linear DNA (genomic) NO NON-terminal CDS 1..15 10 TTT TTT TTT TTT TTT 15 Phe Phe Phe Phe Phe 1 5 5amino acids amino acid linear protein 11 Phe Phe Phe Phe Phe 1 5 18 basepairs nucleic acid single linear DNA (genomic) NO NO N-terminal CDS1..18 12 TTT TTT TTT TTT TTT TTT 18 Phe Phe Phe Phe Phe Phe 1 5 6 aminoacids amino acid linear protein 13 Phe Phe Phe Phe Phe Phe 1 5 12 basepairs nucleic acid single linear DNA (genomic) NO NO N-terminal CDS1..12 14 AAA AAA AAA AAA 12 Lys Lys Lys Lys 1 4 amino acids amino acidlinear protein 15 Lys Lys Lys Lys 1 15 base pairs nucleic acid singlelinear DNA (genomic) NO NO N-terminal CDS 1..15 16 AAA AAA AAA AAA AAA15 Lys Lys Lys Lys Lys 1 5 5 amino acids amino acid linear protein 17Lys Lys Lys Lys Lys 1 5 18 base pairs nucleic acid single linear DNA(genomic) NO NO N-terminal CDS 1..18 18 AAA AAA AAA AAA AAA AAA 18 LysLys Lys Lys Lys Lys 1 5 6 amino acids amino acid linear protein 19 LysLys Lys Lys Lys Lys 1 5 12 base pairs nucleic acid single linear DNA(genomic) NO NO N-terminal CDS 1..12 20 CCC CCC CCC CCC 12 Pro Pro ProPro 1 4 amino acids amino acid linear protein 21 Pro Pro Pro Pro 1 15base pairs nucleic acid single linear DNA (genomic) NO NO N-terminal CDS1..15 22 CCC CCC CCC CCC CCC 15 Pro Pro Pro Pro Pro 1 5 5 amino acidsamino acid linear protein 23 Pro Pro Pro Pro Pro 1 5 18 base pairsnucleic acid single linear DNA (genomic) NO NO N-terminal CDS 1..18 24CCC CCC CCC CCC CCC CCC 18 Pro Pro Pro Pro Pro Pro 1 5 6 amino acidsamino acid linear protein 25 Pro Pro Pro Pro Pro Pro 1 5 12 base pairsnucleic acid single linear DNA (genomic) NO NO N-terminal CDS 1..12 26AGT CAG TCA GTC 12 Ser Gln Ser Val 1 4 amino acids amino acid linearprotein 27 Ser Gln Ser Val 1 15 base pairs nucleic acid single linearDNA (genomic) NO NO N-terminal CDS 1..15 28 AGT CAG TCA GTC AGT 15 SerGln Ser Val Ser 1 5 5 amino acids amino acid linear protein 29 Ser GlnSer Val Ser 1 5 18 base pairs nucleic acid single linear DNA (genomic)NO NO N-terminal CDS 1..18 30 AGT CAG TCA GTC AGT CAG 18 Ser Gln Ser ValSer Gln 1 5 6 amino acids amino acid linear protein 31 Ser Gln Ser ValSer Gln 1 5

What is clamied is:
 1. An anhydrous oxidizing iodine-containingcomposition for use in oligonucleotide synthesis comprising acombination of iodine dissolved in glacial acetic acid as firstcomponent and pyridine as second component.
 2. The composition of claim1 wherein the first component consists of a saturated solution of iodinein glacial acetic acid.
 3. The composition of claim 1 wherein the ratiosof the volumes of the two components equals 1.